Sequence regulating the anther-specific expression of a gene and its use in the production of androsterile plants and hybrid seeds

ABSTRACT

The regulatory sequence is useful for the specific expression in anther of a nucleotide sequence of interest, for example, a sequence whose controlled expression under the regulatory sequence causes complete ablation of the anther in the early development stages and allows a user to obtain androsterile plants that are useful for producing hybrid seeds.

RELATED U.S. APPLICATIONS

[0001] Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

REFERENCE TO MICROFICHE APPENDIX

[0003] Not applicable.

FIELD OF THE INVENTION

[0004] Agriculture. The present invention is related with the obtentionof regulating DNA sequences (promoters) and, using said sequences, withthe construction of new chimeric genes able to be specifically expressedin the anthers of transgenic plants. The invention also relates to theproduction of androsterile plants and hybrid seeds.

BACKGROUND OF THE INVENTION

[0005] To provoke the specific expression of certain genes in theanthers of transgenic plants, different promoters have been isolated,characterised and used. The expression of these promoters is specific toa given tissue of the anther. Some of them have been used in cellablation techniques to produce sterile plants of great utility for theproduction of hybrid seeds. Currently, genetic ablation of plant cellsis being used to carry out studies of cell functioning and signallingwithin specific organs or tissues and to generate androsterility(male-sterility).

[0006] Genetic ablation is based on the induction of cell death by meansof the expression of any enzyme that is able to destroy cell integritysuch as proteases, lipases and RNases (for example, barnase and T1RNase). Equivalent results can be obtained by expressing toxicsubstances into the cells [Day C D and Irish V F. Trends Plant Sci., 2:106-111, 1997]. An example of this last method is the use of peptidesthat deactivate ribosomes, as is the case of the A chain of the toxinfrom Corynebacterium diphtheria (DTA) and the exotoxin A fromPseudomonas aeruginosa. Several groups have suggested the production ofandrosterility via overproduction of growth regulators. The synthesis ofauxins and cytokinines using genes of non-plant origin, such as genes 1and 2 of the Ti plasmid, or the genes rol A, B and C of the plasmid Ri,represent methods where these factors become toxins due to the magnitudeand inappropriate moment of expression.

[0007] Methods have also been developed that do not directly destroy thetissue, but rather give rise to cells susceptible to specific ablativeagents. An example of this approach is the use of an “antisense” RNAfrom a previously established gene. This confers inherited resistance toa chemical agent, such as, for example, tolerance to a herbicide(Fabijanski SF et al., In vitro Cell. Dev. Biol. 28: 46-52, 1992]. Theeffect of the “antisense” RNA is to specifically eliminate the chemicalresistance, for example, in pollen, so that the application of theherbicide leads to destruction of the pollen. This method converts aherbicide into a gametocide.

[0008] In order to obtain an efficient genetic ablation, it is crucialto have a cytotoxic gene that acts only where it is expressed and tohave an appropriate promoter that controls the spatial-temporalexpression of the cytotoxic gene. For this reason, the characterisationof active gene promoters in different cell types and/or at differentmoments during differentiation of the anther has allowed to examine thefunctions of different tissues of the anther during the gametogenesis bymeans of cell ablation [Mariani C et al. Nature, 347: 737-741, 1990;Paul W et al., Plant Mol. Biol., 19: 611-622, 1992; Hird D L et al.,Plant J., 4: 1023-1033, 1993], with particular emphasis on the cell tocell interactions [Roberts M R et al., Sex. Plant Reprod., 8: 299-307,1995]. Similarly, this technique is being used by large seed producingcompanies for the development of androsterility, a desirable feature inthe processes for obtaining hybrid seeds [Williams M E, TrendsBiotechnol., 13: 344-349, 1995].

[0009] In several works, the function of cell to cell interactions hasbeen analysed during the development of the reproductive structures. Forexample, several different promoters have been used to direct theexpression of a cytotoxic gene in cells from tapetum in the anther withthe object of determining the effect of ablation on the development ofpollen [Mariani C et al., Nature 347: 737-741, 1990; Roberts M R et al.,Sex. Plant Reprod. 8:299-307, 1995]. Ablation of tapetal cells indifferent studies has different effects on the development of pollen.The use of a specific promoter of tobacco tapetal cells (TA29) directingthe expression of the barnase gene (ribonuclease) during the tetradphase of the pollen development leads to androsterility, which indicatesthat the tapetum is essential for the production of viable pollen atthis stage (Mariani C et al., Nature 347: 737-741, 1990). On thecontrary, the substitution of the TA29 promoter by the APG promoter fromArabidopsis, specific to tapetur in the microspore phase of pollen, doesnot have any effect on the pollen [Roberts M R et al., Sex. PlantReprod. 8:299-307, 1995]. This latter datum indicates that the tapetumis not essential for the formation of pollen from the disintegration ofthe microspore tetrads. A histochemical analysis of the antherdevelopment in transgenic plants of Brassica with the TA29-barnaseconstruct showed the degradation of RNA within the tapetal cells alongwith the disappearance of RNA from the microspores [De Block M andDebrouwer D, Planta 189: 218-225, 1993]. This observation suggests thatthe microspores remain permeable to small molecules after initiation ofthe deposition of sporopollenine and in late phases of theirdevelopment, since the TA29 promoter does not direct the expression ofgenes in microspores.

[0010] Beals T P and Goldberg R B [Plant Cell, 9: 1527-1545, 1997] putinto practice a new cell ablation strategy for determining what celltypes from an anther are implicated in the dehiscence process. Theytransformed tobacco plants with two constructs: the construct formed bythe TA56 promoter, active in the septum, in the stomium and in theconnective tissue, and the barnase gene along with one of the followingconstructs in an alternative form: a) the TP12 promoter, active in mostof the tissues of the anther, along with the barstar gene (barnaseinhibitor), b) the TA20 promoter, active in most of the tissues of theanther but with a different distribution pattern from that of TA12, andthe barstar gene and c) the soybean lectin gene promoter, active in thecells of the connective tissue, the septum and the stomium, in additionto the barstar gene. The analysis of the different phenotypes of thetransgenic plants showed that the dehiscence process only depends on thepresence of a functional stomium.

[0011] Shull [J. Ind. Abst. Vererb. 12: 97-149, 1914] was the first oneto introduce the term heterosis to describe the advantages offered byheterozygosis regarding cell division, growth and other physiologicalactivities of an organism. The result of these advantages: increase insize, vigour, yield, an earlier fructification and resistance todiseases has for some decades induced the attempts to obtain hybridvarieties [Tsaftaris S A, Physiol. Plantarum 94: 362-370, 1995]. Mostplants containing both male and female reproductive organsself-pollinate themselves (corn, rice, soybean, tomato, etc.), whichcauses problems in the processes for producing hybrid seeds [Kriete G etal., Plant J. 9: 809-818, 1996]. To avoid this problem, a system has tobe used that controls the self-pollination. This system may bemechanical, chemical or genetic.

[0012] The mechanical system consists of manually eliminating theanthers from flowers (emasculation), which is an arduous and expensivetask.

[0013] The chemical method is based on the use of chemical products(gametocides) that specifically destroy the pollen leading to transitoryandrosterility. This approximation is not particularly effective incultures with a long blooming period or with variable or uncontrollableblooming conditions. In addition, the commercial production of hybridcells via gametocides is characterised by being expensive and by thehigh relative effectiveness of the chemical products.

[0014] Most commercial systems for the production of hybrid seeds arebased on genetic methods to control the blooming, so that mutuallyincompatible plants or androsterile plants are used, that is, plantsthat do not produce pollen, are unable to release the pollen or developpollen that is unable to product self fertilisation [Homer H T andPalmer R G, Crop Sci. 35: 1527-1535, 1995]. The production ofandrosterile plants is of great utility for obtaining hybrid seeds. Themale sterility eliminates the possibility of self-fecundation of theplant, thus facilitating the production of hybrids that find importantapplications in the genetic improvement programmes.

[0015] The methods for obtaining hybrid seeds described up until nowhave limitations and are not applicable in important cultures ofagricultural interest. Currently, new strategies are being developedbased on genetic engineering to produce androsterile plants [Gates P,Biotechnol. Genet. Engineering Rev. 13: 181-195, 1994]. The developmentof methods based on recombinant DNA and the characterisation of genesimplicated in the development of pollen has allowed the proliferation ofmolecular systems that provoke nuclear male sterility (NMS), [Scott R Jet al., Plant Sci. 80: 167-191, 1991]. As has already been mentioned,the androsterility in molecular systems is achieved by means of cellablation processes preventing the development of the microspores or thetissues that lead to their development (tapetum and walls of the pollensacs). In addition, it is possible to produce completely functionalpollen, but this pollen is not released due to defects in the structureof the anther. To obtain androsterile plants useful in the production ofhybrid seeds it is important to have a specific promoter for the tissuesimplicated in the development of the pollen, a system for selecting theandrosterile line and a system for restoring the fertility in the F1hybrid line.

[0016] Specific Promoters

[0017] The transcription process in most plant genes is controlled bothtemporally and spatially. The regulation of genetic activity is mediatedby the interaction between trans factors and cis regulatory elementspresent in the promoter region of the gene. Thus, a promoter is a DNAsequence that directs the transcription of a structural gene andtherefore is located in its 5′ region.

[0018] The genes that are exclusively expressed in the reproductiveorgans of the flower (stamen and carpels) are particularly interesting,as their promoters would potentially be able to direct the expression ofother genes towards said organs and provoke male or female sterility inthe flower. This is the case in some gene promoters that arespecifically expressed in the anthers, which have already been used, incombination with ablative agents that only affect the developing pollen,in biotechnological approaches that aim to produce androsterile plantsunable to self-pollinate. Therefore, these types of approach greatlydepend on the existence of promoters providing a suitable expression.During the last years, numerous genes have been isolated andcharacterised that are specifically expressed in tissues and cellsrelated to the development of pollen, therefore, promoters are availablefor this end [Scott R J et al., Plant Sci. 80: 167-191, 1991].

[0019] The promoters that are selected for expressing toxic agents orthose that are degenerative for the tissue are usually promotersregulated by development, sufficiently active and specific for thetarget cells. If the target cell is tapetal tissue, the promoter shouldbe active early in the development of the microspore in order to haltthe process before the microspores become independent from the supportof the tapetum. Similarly, the promoter should act before meioticsegregation to prevent the lack of degenerative activity in part of themicrospores if the lethal gene is hemizygotic.

[0020] An alternative use of promoters regulated by development is theuse of inducible promoters. However, the number of described promotersshowing specific chemical induction is very small. The use of aninducible promoter for blocking the development of pollen has advantageswhen it comes to maintaining and increasing the female line because theplants are fertile and can be multiplied by self-crossing. Inheritedactive or inducible promoters can also be used if the androsterility isbased on the suppression of a gene that is expressed in a tissueimplicated in the development of pollen, for example, via “antisense”.

[0021] Selection of the Androsterile Line.

[0022] In order to produce hybrid seeds in industrial quantities it isnecessary to increase the female line. Although it is possible toproduce many androsterile plants by means of in vitro propagation, forthe plants of agricultural interest, this would be too expensive. Acommon strategy for multiplying the androsterile line is to join a genethat confers resistance to a herbicide to the ablative gene [Mariani Cet al., Nature 357: 384-387, 1992]. After crossing the androsterile linewith an isogenic and fertile line, the plants that have not inheritedthe sterility are eliminated by the herbicide. By analogy, any geneallowing discrimination between the two phenotypes could be used, suchas for example those affecting the pigmentation of the seeds.

[0023] Restoring Fertility

[0024] The restoration of fertility in hybrid plants can be carried outby crossing them with transgenic lines expressing an inhibitor specificto the toxic enzyme used for producing the androsterility, as is thecase for the barstar gene, the product of which inhibits the action ofbarnase [Mariani C et al., Nature 357: 384-387, 1992] or an “antisense”RNA of the lethal gene used [Schmülling T et al., Mol. Gen. Genet. 237:385-394, 1993].

[0025] Molecular Systems Used for Obtaining Androsterile Plants

[0026] The first ablation strategy designed for producing androsterilitywas proposed by Mariani et al. [Nature 347: 737-741, 1990]. The promoterof the TA29 tobacco gene, specific to tapetum, was used for directingthe expression of two different RNases (T1 Rnase from Aspergillus oryzaeand barnase from Bacillus amyloliquefaciens) in tobacco and Brassicanapus. The obtained androsterile transgenic anthers lacked the tapetumand contained the pollen sacs with no microspores or pollen grains. TheTA29-barnase construct was fused with the bar gene, a gene that conferstolerance to the ammonium gluphosinate herbicide, to allow selection ofthe androsterile plants in a population. The application of theherbicide to the progeny of a cross eliminates the fertile male plantsand thus increases the efficiency with which the sterile plants can beisolated. Despite all this, these transgenic plants were no better thanthe spontaneous mutants if there was no possibility of reversing theandrosterility. Mariani et al. [Nature 357: 384-387, 1992] solved thisproblem with the production of transgenic plants with a constructcontaining the inhibitor gene of ribonuclease barnase (barstar) underthe control of the TA29 promoter. The TA29-barstar plants act as arestoring line and androfertile plants are obtained when they arecrossed with plants transformed with the TA29-barnasa construct.

[0027] Apart from this system, the literature contains other methods forproducing androsterile lines:

[0028] In Petunia hybrida, the nuclear androsterility was provoked bysuppressing the synthesis of flavonoids in the anther, which preventsthe maturation of the pollen. This was achieved in two different ways:through the “antisense” effect of RNA [van der Meer I M et al., PlantCell 4: 253-262, 1992] and through a co-suppression [Taylor L P andJorgensen R, J. Hered. 83: 11-17, 1992] of the chalconsynthetase gene,an enzyme implicated in the synthesis route of flavonoids. To restorefertility, flavonoids can be applied to the stigma or mixed with thepollen to allow the androsterile line to multiply by self pollinationand therefore, homozygotic lines are obtained for the androsterilephenotype not requiring the use of any marker for selection [Ylstra B etal., Plant J. 6: 201-212, 1994].

[0029] In tobacco, the androsterility has also been induced by theexpression of the gene rol C of Agrobacterium rhizogenes fused with thepromoter CaMV 35S. Unfortunately, the androsterile phenotype wasaccompanied by other phenotypic alterations in the transgenic plant[Schmülling T et al., EMBO J. 7: 2621-2629, 1988]. The restoration ofsterility was carried out by expression of an “antisense” RNA of thegene rol C in the F1 hybrids [Schmülling T et al., Mol. Gen. Genet. 237:385-394, 1993].

[0030] O'Keefe et al. [Plant Physiol. 105: 473-482, 1994] described asystem of inducible androsterility based on the expression of theP450_(SU1) cytochrome in tobacco tapetal cells. This protein is able totransform an inoffensive derivative of the R7402 gametocide, exogenouslyadded, into its active form (500 times more toxic). However, possiblydue to the fast metabolism of R7402, the androsterility is limited toflowers in a certain phase of development during the application of thecompound. In addition, R7402 is itself toxic and begins to inhibitgrowth when it is applied in quantities four times greater than thoseused to produce androsterility in the classical way.

[0031] Another system for obtaining inducible androsterility is based onthe use of the TA29 promoter of tapetum along with the argE gene from E.coli. The product of this gene deacetylises theN-acetyl-L-phosphinotrycin compound and transforms it into gluphosinate,a cytotoxic compound. When the N-acetyl-L-phosphinotrycin is applied tothe tobacco plant, the tapetum degenerates and androsterile plants areobtained [Kriete G et al., Plant J. 9: 809-818, 1996]. Finally, we wouldlike to point out that androsterility, in addition to being an importanttool for obtaining hybrids, is also a desirable feature in plantscapable to develop fruits in the absence of fertilisation (partenocarpicfruits). In this type of fruit, the seeds are absent and so theconsumption or acceptance thereof by the consumers is increased [RotinoG et al., Nat. Biothecnol. 15: 1398-1401,1997]. Some partenocarpiccultures of agricultural interest are: pears, citric fruits, cucumber,grape and dates.

BRIEF SUMMARY OF THE INVENTION

[0032] In general, the invention addresses the problem of developing asystem useful for producing androsterility in plants.

[0033] The solution provided by this invention is based on the isolationand characterisation of a promoter able to direct the specific antherexpression in early development phases of the plant, in particular, theEND1 gene promoter from pea (Pisum sativum L.). The use of said promoterallows to produce transgenic plants that express a gene specific to theanther, for example, a gene that provokes the ablation of the anther andtherefore gives rise to an androsterile plant, of greater use in geneticimprovement programmes for obtaining hybrid seeds. In Example 1, theproduction of transgenic plants containing different constructionscomprising the END1 promoter fused to a reporter gene is described,observing its specificity on directing the expression of the reportergenes in anthers. Example 2 describes the production of androsterileplants of Arabidopsis thaliana by using the barnase gene through theEND1 promoter. The obtained results show that said construct provokesthe complete ablation of anthers from very early stages in theirdevelopment, preventing the formation of pollen therein and leading tomale sterility of the plant with a 100% effectiveness.

[0034] Accordingly, an object of this invention is a sequence ofnucleotides regulating the specific expression in the anther of a genecomprising the nucleotide sequence shown in SEQ ID NO 1, or a fragmentthereof, or a nucleotide sequence substantially homologous to saidsequences. The use of said nucleotide sequence for the production ofandrosterile plants and hybrid seeds constitutes an additional object ofthis invention.

[0035] Another additional object of this invention constitutes a DNAconstruct comprising the whole or part of said nucleotide sequence, aswell as a vector containing said sequence or DNA construct and a celltransformed with said vector.

[0036] Another additional object of the invention constitutes the use ofsaid DNA sequence, or of said DNA construct, in the production oftransgenic plants that specifically express in anthers a DNA sequence ofinterest, for example, transgenically androsterile plants that express acytotoxic polypeptide or RNA, the expression of which induces theablation of the anther. The resulting transgenic plants constituteanother additional object of this invention.

[0037] Another additional object of this invention constitutes a methodfor producing hybrid seeds comprising introducing into said DNAconstruct into a plant.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0038]FIG. 1 shows the space-temporal analysis of the expression of END1in longitudinal sections of pea flowers in different phases. The purplecoloured precipitate indicates the site of expression. A. Floral shootin which the common primordia (s) have still not differentiated intopetaloid and staminal features along with a flower on day 12 before theanthesis where the END1 begins to be expressed. B. Flower on day 10before anthesis. C. Control of the in situ hybridisation: sectionshybridised with “sense” riboprobe. D. Flower on day 8 prior to anthesis.E and F. Flowers on day 6 prior to anthesis photographed with smallerand greater magnification, respectively. G and H. Serial sections from aflower on day 5 prior to anthesis hybridised with the “sense” riboprobe(control) and “antisense” riboprobe, respectively. C, carpel; E,stamens; P, petals; Co, connective tissue; M, microspores; T, tapetum;1, 2, 3 and 4, whorls 1, 2, 3 and 4 of the flower.

[0039]FIG. 2 shows the sequence of the promoter region of END1. The twopossible TATA boxes (tataaat and tatatata), a CAAT box and two CCAATboxes appear underlined. In blue, three possible CarG boxes arerepresented, in green a GTCAAAA motif, an element ACGTCA in pink andthree motifs C(A)_(6/8) in red. The GTCAAAA box shares its last fivenucleotides with one of the boxes C(A)_(6/8). The annotation +1indicates the cDNA sequence of the clone 162. The translation initiationcodon (ATG) is shown in boldface. In addition, there is shown thededuced amino acid sequence. The nucleotides in boldface indicate thecodon for stopping translation (taa) and the possible signal ofpolyadenylation (aataaa).

[0040]FIG. 3 shows the stacking of the consensus sequence of joining toDNA of AGAMOUS (CArG box and adjoining sequences) with a putativeequivalent sequence found in the promoter region of END1.

[0041]FIG. 4 shows a schematic representation of the pBI101-F3 andpBI101-F1.5 constructs used for transforming plants of Arabidopsis,tobacco and tomato. The plasmid pBI101 used for making the constructsconsists of: the inherited promoter of nopaline synthetase (nos-pro)fused to the nptII gene which confers resistance to kanamycin, the uidAgene which codes for the enzyme b-glucuronidase (GUS-intron) and thepolyadenylation signal of the nopaline synthetase gene (nos-ter) at the3′ extremes of both genes.

[0042]FIG. 5 shows the results of expression of the pea END1 promoterfused with the uidA gene (GUS-intron) in transgenic plants ofArabidopsis thaliana. A and B: After performing the correspondinghistochemical assays of b-glucuronidase activity (GUS) with flowers indifferent developmental phases, said activity was only detected in theanthers (blue) and never in other floral organs or in the rest of theplant. C: Longitudinal section of a stamen with GUS activity included inparaffin. The GUS activity (blue) is detected in cells conforming theepidermis, the connective tissue and the endothecial tissue. No GUSactivity was detected either in the tapetum or in the pollen. Althoughin the figure some grains of pollen appear blue, this is an artefact, asthe blue colour only appears on the exine coating and never inside. Co:connective, En: endothecial, Ep: Epidermis.

[0043]FIG. 6 shows the results of expression of the pea END1 promoterfused with the uidA gene (GUS-intron) in transgenic tobacco plants(Nicotiana tabacum). A, B and C: Histochemical assays of GUS activity intobacco flowers in different development phases showing activity only inthe anthers (blue). D: Fresh transversal section of an anther showingthe pollen grains and the remaining tapetum tissue with no bluecolouring. E: stamens, C: carpel, P: petals, S: sepals, Po: pollen.

[0044]FIG. 7 shows the results of expression of the pea END1 promoterfused with the uidA gene (GUS-intron) in transgenic tomato plants(Lycopersicom sculentum). a: Histochemical assays of GUS activity intomato flowers showing activity specifically in the anthers (St). b:Section in paraffin of one of these flowers, the GUS activity isrestricted to tissues that form the pollen sacs (epidermis, endothecium,connective, etc) but do not exist in the tapetum or in the pollen. Se:sepals, Pe: petals, St: stamens. Ca: carpel, Ep: epidermis, Co:connective, En: endothecium, Tp: tapetum and Po: pollen.

[0045]FIG. 8 shows the results of transgenic Arabidopsis thaliana plantswith male sterility by using the BARNASE gene directed by the END1promoter. a: Control plant of Arabidopsis (WT) showing the siliques(arrows, right box) formed as a result of the fertilisation of itsovaries with mature pollen. Beside it, a transgenic plant can be seen inwhich the effects of expression of the barnase gene under the control ofthe END1 promoter is shown: The ageing ovaries (right box) remain in theplant as they have not been pollinated (arrows). b: Normal flower of awild type plant of Arabidopsis after pollination of the stigma by meansof dehiscence of the anthers (phase 14). After the maturing process ofthe anther, the filament has undergone a lengthening until the pollensacs are located at the height of the stigma to facilitate theirpollination. c: Flower of a transgenic plant in the same state showingthe BARNASE effects on the development of the anther. The ablation ofthe anther is complete, with no pollen sacs being formed. The filamentdoes not undergo the lengthening process, as the development of theanther is not completed. In these conditions, pollination is impossibleand the plant is 100% sterile.

[0046]FIG. 9 shows the result of studies by means of a scanning electronmicroscope (SEM) of the cell types present in the structures formed inthe place of the anthers of transgenic plants bearing the pEND1-barnaseconstruct. a: Stamen of a control flower of Arabidopsis. The black arrowindicates the cell types present in the epidermis of the anther(saw-tooth edges) and the black one indicates those of the filament(lengthened). b: The same stamen seen from the opposite side. A changein cell type is observed (more rounded) in the area where the filamentreach the anther (arrow). c: Stamen of a transgenic plant, the celltypes present in the filament are observed and the rounded ones at theinsertion zone, but not those of the epidermis of the pollen sacs. d:Detail of the structure present in the terminal zone of the filament inwhich the lengthened cell types of the filament coexist with the roundedtypes of the insertion zone.

[0047]FIG. 10 shows the result of histochemical studies in paraffinsections of the structures formed in place of the anthers in transgenicplants bearing the pEND1-barnase construct. a: Longitudinal section of acontrol flower of Arabidopsis in an early development stage of theanther. In red, a group of mother cells of the microspores can be seenthat will give rise to the mature pollen after successive divisions. b:Control anther in a more advanced developmental stage (12-13, beforedehiscence) in which the almost complete disappearance of the tapetumcan be observed and the pollen is found in a maturing process (red). Thefilament has begun to lengthen. c and d: Longitudinal sections of twotransgenic flowers in stage 13 and 12 respectively, in which thestructures formed in place of the stamens are observed. Inside thewidening produced in the terminal zone, a small group of pollen mothercells is observed and maybe someone of the external tapetum (red) thathave stopped their development (there are no divisions). The filamenthas not begun to lengthen. e: Detail of one of the structures shown infigure d where detail is shown of the group of round pollen mother cellsand possible tapetal cells.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The invention provides a sequence of nucleotides regulating thespecific expression in the anther of a gene, hereinafter the nucleotidesequence of the invention, selected from:

[0049] a) A nucleotide sequence comprising the nucleotide sequence shownin SEQ ID NO 1;

[0050] b) A fragment of said SEQ ID NO 1 retaining its capacity toregulate specific expression in the anther; and

[0051] c) A nucleotide sequence that is substantially analogous to thesequence of nucleotides defined in a) or in b).

[0052] In the sense used in this description, the term “analogous” is toinclude any sequence of nucleotides having at least capacity to regulatespecific expression in the anther. Typically, the analogous DNAsequence:

[0053] Can be isolated from any organism producing said analogoussequence based on the nucleotide sequence shown in SEQ ID NO 1, or

[0054] It is constructed on the basis of the nucleotide sequence shownin SEQ ID NO. 1 by means of substitution of one or more nucleotides, theinsertion of one or more nucleotides in the sequence, the addition ofone or more nucleotides at either end of the sequence, or the deletionof one or more nucleotides at either end or in the inner part of thesequence. For example, the analogous DNA sequence can be a subsequenceof the nucleotide sequence shown in SEQ ID NO 1.

[0055] In general, the analogous DNA sequence is substantiallyhomologous to the nucleotide sequence identified as SEQ ID NO 1. In thesense used in this specification, the expression “substantiallyhomologous” means that at a nucleotide level the nucleotide sequences inquestion have an identity degree of at least 60%, preferably at least85%, or more preferably at least 95%.

[0056] The nucleotide sequence of the invention can originate from anyorganism that contains it, for example, from pea (Pisum sativum L.) orfrom a host organism transformed with said DNA sequence. The nucleotidesequence of the invention can be isolated by means of conventionaltechniques, starting from the DNA from any other species by employingoligonucleotide probes prepared from information on the nucleotidesequence of the invention.

[0057] In a particular embodiment, the nucleotide sequence of theinvention is the sequence shown in SEQ ID NO 1, corresponding to thenucleotide sequence of the promoter of the END1 gene of pea (Pisumsativum L.).

[0058] In previous works carried out in our laboratory, we isolated andcharacterised a gene of the pea (Pisum sativum L.) that was exclusivelyexpressed in certain tissues of the anthers. Its isolation andcharacterisation was carried out by means of the prior purification andidentification of the protein it codes. To do this, a process ofimmunosubtraction of extracts from stamens was carried out withpolyclonal serum obtained by immunisation of a rabbit with extracts fromthe other floral organs (sepals, petals and carpels). The result of thisprocess was the production of an extract enriched in proteins specificto the stamens, eliminating those that these organs have in common withthe remaining floral organs. Said enriched extract was used to immunisemice and obtain hybridoma lines that produced monoclonal antibodiesspecific for a certain staminal protein. One of those (MAbA1) recogniseda protein of 26 kDa (END1) present in much abundance in the stamensextracts and whose immunolocalisation indicated to us that itaccumulated exclusively in the tissues that make up the architecture ofthe pollen sacs of the anther. Using affinity chromatography, wepurified a certain amount of said protein and sequenced 20 aas of itsN-terminus. The obtained sequence had a high homology with anotherprotein (PA2) previously described in pea cotyledons and whose functionis unknown [Higgins et al., Plant Mol. Biol. 8: 37-45, 1987]. The use ofthe cDNA of PA2 as a probe in the screening of a gene library of peaflowers allowed us to isolate a clone with the complete coding sequence(910 bp, which we call END1, SEQ ID NO 2), which shows 72% homology withthat of PA2. The Northern Blot assays showed the specificity ofexpression of this new protein as it was restricted to anther extractsand was not detected in other floral organs, cotyledons and planttissues. On the other hand, the in situ hybridisation assayscorroborated the specificity of the expression of the END1 gene in theanther tissues of the pea during the different developmental phases(FIG. 1). Its expression started in early developmental phases(differentiation of the common primordia cells in petals and stamens)and continued until the dehiscence of the anther, being expressed onlyin the epidermis, connective tissue, middle layer and endothecium.Expression of the gene was not detected in the nutritive tissue(tapetum) or in the germinal tissue (pollen). The assays carried outwith other floral organs, other parts of the plant (stem, leaves, roots,etc.) or with seeds (cotyledons) were negative.

[0059] The specific expression of the END1 gene in those tissues thatform the pollen sacs of the anthers suggested to us the isolation andanalysis of the promoter of said gene. The study of this promoter wasinteresting for different reasons. First, we could determine whichmotifs of a promoter determine the specific expression of a gene in theanther. Similarly, it would be interesting to analyse whattranscriptional factors regulate the expression of the END1 gene and todetermine whether these are related to the MADS-box homeotic genes whichregulate the identity of the floral organs, in particular, with genes ofclass B and C that are implicated in the development of stamens. Withthese objectives, we examined a genomic DNA library of the pea, usingthe complete fragment of the END1 cDNA (SEQ ID NO 2) as a probe. Aninitial examination was carried out with 500,000 phage plaques in whichwe found a positive clone (clone 162). This was purified and isolatedafter three more examinations. By means of the culture and positivephage lysis we purified its DNA. This DNA was digested with HindII, asthis enzyme spliced the fragment of cDNA at 210 bp from its start andfrom the phage digestion we could obtain a DNA fragment containing thefirst 210 bp of cDNA plus more promoter sequences upstream from these.With the digested DNA, a Southern Blot analysis was carried out usingthe 210 bp of the 5′ terminus of the cDNA fragment of the clone 162 as aprobe to hybridise. This fragment was used as a probe to determine whichfragment resulting from the restriction would be the one constituted bythese 210 bp and part or all of the promoter sequence. The Southern Blotanalysis showed that a fragment of approximately 3 Kb derived from thedigestion of phage DNA with HindII hybridised with the probe. Thefragment of 3 Kb was purified using the QUIAEXII method, cloning in theBluescript-KS(−) vector at the HindII site of the cloning site andsubsequently sequenced. The complete nucleotide sequence of the END1promoter is represented in FIG. 2 and in SEQ ID NO 1 (the nucleotidesfrom −2736 and 1 of FIG. 2 correlate with the nucleotides 1 and 2766 ofSEQ ID NO 1, respectively).

[0060] After sequencing the cloned fragment, we observed that we haveisolated a fragment of 2946 bp. Of these, the 210 bp of the 3′ terminuscorresponded to the first 210 bp of the fragment of the clone of cDNA162. At positions −263 (SEQ ID NO 1, 2474) and −56 (SEQ ID NO 1, 2681),taking the first of the isolated cDNA 162 as the nucleotide +1 (in SEQID NO 1 the nucleotide +1 corresponds to nucleotide 2377), two possibleTATA boxes, at positions −347 (SEQ ID NO 1, 2390) and −66 (SEQ ID NO 1,2671), two putative CCAAT boxes, and at position −401 a CAAT box (SEQ IDNO 1, 2336) (FIG. 2) are found. In addition to these boxes, common toeukaryotic promoters, boxes typical of plant promoters have also beenfound. In Table 1 all the motifs of the plant promoters found betweennucleotide −400 (SEQ ID NO 1, 1977) and +1 from the promoting region ofEND1 are defined using the database of the National Institute forAgro-biological Resources of Japan. We select this area on the basisthat the distances at which the functional boxes are found in thepromoters with respect to the start of transcription do not normallyexceed 400 nucleotides according to references in the scientificliterature on the different boxes studied. TABLE I Description of thecis elements localised in the fragment lying between the nucleotides−400 and −1 of the promoter region of end1. POSITION CONSENSUS MOTIF(CHAIN) SEQUENCE REFERENCE ELEMENT −300 −157 (+) TGHAAARK Thomas andFlavell, 1990 AP3 −209 (+) TGTGGWWW Mercurio and Karin, 1989 ASF1 −122(−) TGACG Terzaghi and Cashmore, 1995 G-BOX −84 (+) CACGTG Foster etal., 1994 E-BOX −333, −295, −30 (+) CANNTG Kawagoe et al., 1994 GATA−265, −205 (+) GATA Gilmartin et al., 1990 GT1 −278, −156 (+) GRWAAWLawton et al., 1991 HEXMOTIF1 −127 (+) ACGTCA Mikami et al., 1989 I-BOX−356 (−), −194 (−) GATAA Giuliano et al., 1988 MYB-PH3 −234 (+) CNGTTASolano et al., 1995 MYB-ST1 −206 (+) GGATA Baranowskij et al., 1994ROOTMOTIF1 −202 (+), −96 (+) ATATT Elmayan and Tepfer, 1995 RY −34 (+)CATGCAY Fujiwara and Beachy, 1994 SIF −215 (+) ATGGTA Zhou et al., 1992SEF1 −392 (+) ATATTTAWW Lessard et al., 1991 SEF4 −276 (+) RTTTTTRLessard et al., 1991 MYB-P −349 (+) CCAACC Grotewold et al., 1994 SBF1−150 (+) TTAA Lawton et al., 1991 CarG −103 (−) CC(A/T)₆GG Shiraishi etal., 1993

[0061] Further analysis of the promoter region sequence allowed todetect the presence of other regulatory motifs. Two of them are CArGboxes (⁻³²⁹TGAAAATACC⁻³²⁰ (SEQ ID NO 1 ²⁴⁰⁸TGAAAATACC²⁴¹⁷) and⁻³⁰⁰GGTTTCAACT-⁻²⁹¹ (SEQ ID NO 1 ²⁴³⁷GGTTTCAACT²⁴⁴⁶)), which, althoughnot as similar as the first one to the consensus sequence, can also actas such. An example of CarG boxes that are functional, althoughdiffering from the consensus sequence, is represented by two of thethree CarG boxes (CCTTTCATGG and CCATTTTTAG) of the promoter of the AP3gene of Arabidopsis (Tilly J J et al., Development 125: 1647-1657,1998). Other motifs that we have identified are ⁻²⁹⁰GTCAAAA⁻²⁸⁴ (SEQ IDNO 1 ²⁴⁴⁷GTCAAAA²⁴⁵³) present in the genes Zm13 and LAT52 (Zou J T etal., Am. J. Bot. 81: 552-561, 1994), the motif ⁻¹²⁷ACGTCA-⁻¹²² (SEQ IDNO1 ²⁶¹⁰ACGTCA²⁶¹⁵) localised on the gene Bp19 (Zou J T et al., Am. J.Bot. 81: 552-561, 1994] and the element C(A)_(6/8) repeated three times(at positions −507 (SEQ ID NO 2230-2236), −288 (SEQ ID NO 2449-2457) and−247 (SEQ ID NO 2490-2496)) on the promoters of the genes OlnB4 andOlnB19 [Hong H P et al., Plant Mol. Biol. 34: 549-555, 1997].

[0062] The different motifs found in the promoter region of END1 can begrouped in function of the type of genes where they have been described.The motifs SEF1, SEF4, E-box, RY and Element −300 are characterised bybeing present in gene promoters that code reserve proteins of the seed.The motifs GATA, GT1, I-box and ASF-1 are present in genes whoseexpression is regulated by light. The boxes that recognise factorshomologous to Myb animal proteins: MYB-P, MYB-PH3 and MYB-ST1 have beenfound in genes implicated in the biosynthetic route of phenylpropanoids.The G-box motif is localised on a multitude of plant genes regulated bydifferent environmental and physiological factors. SBF1 has beendescribed on the defence gene chs15 of Phaseolus vulgaris. Rootmotif1has been identified in the promoter sequence of two genes of root ofcorn rolD and pox1. And the elements GTCAAAA, ACGTCA and C(A)_(6/8) arepresent in anther specific genes of Brassica napus, such as Bp19 [Zou JT et al., Am. J. Bot. 81: 552-561, 1994; Hong H P et al., Plant Mol.Biol. 34: 549-555, 1997]. All these motifs are indicated in SEQ ID NO 1.

[0063] Finally, the motif CArG is a DNA consensus sequence that isrecognised by the MADS domain that characterises the homeotic genes ofplants implicated in the development of floral organs [Huang H et al.,Nuc. Acids Res. 21: 4769-4776, 1993; Shiraisi H et al., Plant J. 4:385-398, 1993]. The genes APETALA-1 [AP]; Mandel M A et al., Nature 360:273-277, 1992), APETALA-3 [AP3, Jack T et al., Cell 68: 683-697, 1992),PISTILLATA [P1, Goto K et al., Genes Dev. 8: 1548-1560, 1994) andAGAMOUS (AG, Yanofsky M F et al., Nature 346: 35-39, 1990] are homeoticgenes that regulate the identity of the floral organs in Arabidopsis.These genes belong to the family of proteins containing a MADS domain intheir sequence. The MADS domain is composed of 56 amino acids and isimplicated in the dimerisation of the MADS genes with themselves or withother MADS genes and the subsequent binding to DNA of these dimers[Shore P and Sharrocks A D, E. J. Biochem. 229: 1-13, 1995].

[0064] The type B homeotic genes (AP3 and PI in A. thaliana) control thedevelopment of petals and stamens, and those of type C (AG in A.thaliana) control the formation of stamens and carpels. The END1 gene isexpressed in stamens and, therefore, could be a target gene for the typeB and C MADS genes. This hypothesis is supported by the existence ofthese putative CArG boxes in the promoter region of END1. Huang et al.[Nuc. Acids Res. 21: 4769-4776, 1993] and Shiraisi et al. [Plant J. 4:385-398, 1993] determined that the AG gene would require some consensussequences adjoining the CarG box for binding to DNA. The consensussequence of binding to DNA for the MADS domain of AG was identified as:5′-TT(A/T/G)CC(A/T)₆GG(A/T/C)AA-3′ according to Shiraisi et al. [PlantJ. 4: 385-398, 1993] and TT(A/T)CC(A/T)(A/t)₂(T/A)NNGG(-G)(A/t)₂according to Huang et al. [Nuc. Acids Res. 21: 4769-4776, 1993]. Thesequences adjoining the CArG box at position −103 in the promoter ofEND1, with the exception of two nucleotides, fit with those recognisedby the MADS domain of the AGAMOUS gene elucidated by the previousauthors (FIG. 3). This fact would confirm the hypothesis that END1 couldbe a direct target of a floral homeotic gene, specifically, it could bethe target of the AGAMOUS gene.

[0065] In order to confirm whether the promoting sequence cloned fromEND1 directed the specific expression of a gene in anthers and whetherit was able to do so in plants other than the pea (P. sativum), wetransformed tobacco plants (Nicotiana tabacum), Arabidopsis thaliana andtomato (Lycopersicom sculentum) with the promoter sequence fused to thecoding sequence of the gene of b-glucuronidase (uidA, GUS-intron)[Example 1]. Two different constructs were carried out using the plasmidpBI101: pBI101-F3 and pBI101-F1.5 (FIG. 4). The first construct containsthe nucleotide sequence comprised from nucleotide −2736 and −6 (SEQ IDNO 1, 1 to 2731) and the second one contains the sequence from theresidue −1531 to −6 (SEQ ID NO 1,.1206 to 2731). This latter constructwas assayed to mark, in an initial attempt, the sequence constitutingthe promoter region in the cloned fragment. Both constructs wereintroduced into Arabidopsis using strain C58 of Agrobacteriumtumefaciens, and the construct pBI 101-F3 into tobacco and tomato bymeans of the LBA4404 strain of A. tumefaciens. All these plants weretransformed in turn with the plasmid pBI 101 as a control and theexpression of the reporter gene was subsequently analysed in differenttissues.

[0066] The results of the transformations described in the previousexamples show that the promoter sequence isolated from the END1 gene isfully functional as it directs the heterologous expression of a gene ina specific manner in anthers of plants other than the pea. Moreover,this capacity of the nucleotide sequence lying between position −1531and −6 of the promoter (SEQ ID NO 4) has been described. On the otherhand, the minimum nucleotide sequence that would substantially maintainthis capacity for regulating specific expression in anthers can bedefined with similar experiments. Thus, they form a part of the presentinvention all those nucleotide sequences, either of the complete regionof the END1 promoter or any fragment thereof that substantiallymaintains this capacity for directing the specific expression inanthers. On the other hand, starting form this sequence of the promoterof the END1 gene of the pea, the promoter region of the two geneshomologous with END1 can be obtained in other plant species thanks tothe general knowledge available on molecular biology techniques. Allthese nucleotide sequences homologous with the one described in thepresent invention and which substantially maintain the capacity toregulate the specific expression in anthers form part of the presentinvention.

[0067] These regulatory sequences can be used in the development of DNAconstructs that also include gene coding sequences of interest that inturn can be integrated into recombinant expression vectors. To do this,different techniques can be used that are well known in the state of theart [Sambrook et al, “Molecular cloning, a Laboratory Manual”, 2^(nd)ed., Cold Spring Harbor Laboratory Press, N.AND., 1989 Vol 1-3], some ofwhich are shown in the present invention. These recombinant vectorsallow the expression of different peptide, polypeptide, protein or RNAcoding genes that, as described in the state of the art of the presentinvention, on being toxic for the tissues where they are expressed,would induce the ablation thereof and in this case would lead toandrosterility of the resulting plant. Examples of these genes are thosedisclosed in the European patent application EP 412006 and which can betaken as a reference along with those described in the state of the artof the present invention. On the other hand, these constructs maycontain other elements regulating the specific expression in anthersdepending on the recombinant vector used, the plant to be transformed,etc. All these possible DNA constructs containing as the commondominator the nucleotide sequence of the invention as well as the usethereof for the production of androsterile plants form part of thepresent invention.

[0068] Therefore, the invention provides a DNA construct that comprisesthe nucleotide sequence of the invention and a coding nucleotidesequence for a peptide, polypeptide, protein, activity or RNA ofinterest. In a particular embodiment, said DNA construct comprises acoding sequence of activity promoting the sterility in plants under thecontrol of the nucleotide sequence of the invention. In anotherparticular embodiment, said DNA construct comprises a coding sequence ofan activity that reverse or recovers the fertility of a plant, such as acoding sequence of an activity which is capable to inhibit the promotingactivity of sterility in plants, under the control of the nucleotidesequence of the invention. Said DNA construct can also contain someregulatory elements, operationally linked, to the specific expression inanthers, for example, a terminal sequence of the transcription,depending on the recombinant vector used, the plant to be transformed,etc.

[0069] The sequence of nucleotides of the invention, or the DNAconstruct provided by this invention, can be inserted into anappropriate vector. Therefore, the invention also refers to a vector,such as an expression vector, that comprises said sequence ofnucleotides of the invention, or said DNA construct. The choice ofvector will depend on the host cell in which it is to subsequently beintroduced. By way of example, the vector where said DNA sequence isintroduced can be a plasmid or a vector that, when introduced into ahost cell, is integrated into the genome of said cell and is replicatedalong with the chromosome (or chromosomes) in which it was integrated.To obtain said vector, conventional methods can be used that are wellknown to those skilled in the art [Sambrok et al., 1989, cited earlier].

[0070] The invention also provides a cell that comprises a nucleotidesequence of the invention, or a DNA construct containing said nucleotidesequence or said vector mentioned hereinabove. The host cells that canbe transformed with the sequence of nucleotides of the invention can beprokaryotic cells or, preferably, eukaryotic cells, such as cells ofplant tissue. The transformation of cells of plant tissue can also becarried out using conventional methods. For a review of the genetictransfer to plants, including vectors, methods of DNA transfer, etc, seefor example the book titled “Ingeniería genética and transferenciagénica”, by Marta Izquierdo, Ed. Pirámide (1999), in particular, chapter9, titled “Transferencia génica a plantas”, pages 283-316.

[0071] The nucleotide sequence of the invention can be used fortransforming plants and obtaining transformed plants. The transformationof plants is extensively described in the state of the art. As is wellknown, multiple systems can be used, for example, plasmid vectors,liposomes, electroporation, microinjection, diffusion, gene gun,co-precipitation with calcium phosphate, use of viral vectors, etc. Inthe present invention, the transformation of plants using a plasmidvector is described as an example of the many technical possibilities,all of which form a part of the present invention. In this sense, inExample 1, the transformation of tobacco plants (N. tabacum), tomato (L.sculentum) and Arabidopsis thaliana are described as examples of planttransformation.

[0072] The nucleotide sequence of the invention can be used to regulatethe specific anther expression of a peptide, polypeptide, protein,activity or RNA sequence of interest in a plant. In a particularembodiment of the invention, the nucleotide sequence of the inventioncan be used to produce plants with male sterility (androsterility), thatis, androsterile plants, which comprises transforming a plant with a DNAconstruct provided by this invention, said DNA construct comprising acoding sequence of an activity promoting sterility in plants, forexample, the sequence of a cytotoxic gene, such as a gene that codes aribonuclease activity, under the control of the nucleotide sequence ofthe invention, so that the expression of the coding sequence containedin said construct provokes the complete ablation of the anthers fromvery early developmental stages, preventing the formation of pollentherein and producing androsterility in the plant with 100%effectiveness. The resulting androsterile plant can be used for theproduction of hybrid seeds by means of a method comprising its cultureunder conditions that allow is development and maturing.

[0073] The invention also provides a method for restoring the fertilityof an androsterile plant that comprises transforming said androsterileplant with a DNA construct comprising a coding sequence of an activitythat reverses or restores the fertility under the control of thenucleotide sequence of the invention so that a fertile plant isobtained. In a particular embodiment of the invention, theandrosterility is due to the expression of the barnase gene and therecovery of fertility is due to the expression of a sequence coding anactivity that reverses or restores the fertility such as the expressionof barstar, an activity able to. inhibit the ablation activity of. theanthers caused by BARNASE.

[0074] The plants susceptible to being transformed by the use of thenucleotide sequence of the invention can be both monocotyledonous plantsand dicotyledonous plants, for example, monocotyledonous ordicotyledonous plants of agricultural interest, such as cereals,horticultural plants, e.g., tobacco, tomato, melon, watermelon,cucumber, etc.

[0075] In another particular embodiment, the invention provides atransgenic cell that comprises a nucleotide sequence of the inventionintegrated into its genome, as well as a transgenic plant comprising atleast one of said transgenic cells. Said transgenic plants, whichconstitute an additional object of the invention, can be obtained bymeans of conventional techniques, for example, through the use ofconventional antisense mRNA techniques and/or overexpression (in sensesilencing) or others, for example, using binary vectors or other vectorsavailable for the different plant transformation techniques currently inuse. Examples of transgenic plants forming part of the present inventioninclude both monocotyledon and dicotyledonous plants.

[0076] The nucleotide sequence of the invention is useful for obtainingandrosterile plants and for producing hybrid seeds as it directs theexpression of genes that code for certain enzymes that produce cellularablation in those tissues that form the pollen sacs, as was explainedearlier. This system, in combination with another similar one, whichrestores the fertility on producing an inhibitor of the enzyme used forprovoking cellular ablation, would be of great utility in geneticimprovement programmes based on the production of hybrids (heterosis).With an identical aim, different promoters had been tried before, but a100% sterile plant population has not been attained with all of them[Zhan X et al., Sex. Plant Reprod.9: 35-43, 1996; Roberts M R et al.,Sex. Plant Reprod. 8: 299-307, 1995]. This phenomenon is due to the factthat these promoters are activated in late stages of development whenthe pollen is already practically developed, so that the tissue thatundergoes ablation (tapetum) is no longer strictly necessary for thepollen to reach full maturity. This methodology has a serious problem inthat the final result obtained is a mixed population of hybrid andnon-hybrid seeds. The nucleotide sequence of the invention constitutesan alternative to said promoters because it is activated when thedifferentiation of the staminal primordial cells is initiated. In thisearly phase, a proteinase or a nuclease would impede the correctdevelopment of the anther by destroying the cell lines that will giverise to tissues that form the pollen sacs at an early phase. The tissueswhere END1 is expressed are tissues that provide the support and thestructure (architecture) for the anther, and so it is hard to imagine ananther able to produce pollen without these tissues. In this sense,Example 2 describes the production of androsterile plants of A. thalianawith a 100% efficacy. It is to be reasonably expected that thenucleotide sequence of the invention, in particular the END1 promoter,will be able to maintain its specificity in different plants, bothdicotyledonous and monocotyledonous, as in several of these plants, theMADS genes of class C have been identified (homologous to AGAMOUS) whichcould activate said promoter like the dicotyledons, and so it is to beexpected that said nucleotide sequence of the invention will be usefulfor producing androsterility, both in dicotyledonous plants and inmonocotyledon plants of agricultural interest.

EXAMPLE 1

[0077] Functional studies of the END1 promoter in transgenic plants ofArabidopsis thaliana, tobacco (Nicotiana tabacum) and tomato(Lycopersicom sculentum)

[0078] 1.1—Design of the pBI101-F3 and pBI101-F1.5 Constructs

[0079] The promoter region of the END1 gene was amplified, using the PCRtechnique, from the genomic fragment of DNA cloned in the pBluescriptKS(+) plasmid, using the TB1, TB2 and TB3 oligos (Table 2). TB1introduces a restriction site BamHI, and TB2 and TB3 introduce arestriction site SalI. Two fragments are amplified: one of 2731 bpcontaining almost all the isolated promoter region(−2736/−6) and anotherone of 1526 bp next to the coding region of the gene (−1531/−6 inFIG. 1) (SEQ ID NO 4). Neither of these two fragments contained codingsequence. Both fragments were cloned in the plasmid vector PCR_(2,1).[Clar, J. M. (1998) Nuc. Acids. Res. 16:9677-9686; Mead, D., et al.(1991) Bio Technology 9:657-663]. Subsequently, the cloned inserts werereleased with the restriction enzymes BamHI and SalI, and were cloned inthe plasmid pBI101 directing the expression of the gene uidA(GUS-intron) of b-glucuronidase (Vancanneyt G et al., Mol. Gen. Genet.220:245-250, 1990). At the end of the process, two different constructswere attained: pBI101-F3 and pBI101-F1.5. The first one contained thefragment of 2731 bp and the second one the fragment of 1526 bp (FIG. 4).TABLE 2 Primers used in the PCR amplifications. Primers Sequence(5′ ® 3′) DNA mould TB1 GAGAGCCTAGGAAGGTTATGTTGTGAGC clone F3 TB2GACTCGAGGTCGACTTCAACCTTATTAGTG clone F3 TB3GACTCGAGGTCGACAACCAGTGTGCATATATC clone F3

[0080] 1.2.—Transformation of Arabidopsis thaliana, Nicotiana tabacumand Lycopersicom sculentum

[0081] In order to obtain the transgenic plants of tobacco, two cvs ofN. tabacum were used: Samsun and Pettit Habane SRI. The transformationwas conducted with leaf disks in co-culture with A. tumefaciens (strainLBA4404) for three days at 24° C. in darkness in MSS medium (Marashigeand Skoog Medium 4.4 g/l, sucrose 2%, Mes 100 mg/l, phytagel 3.5 g/l, pH5.9) following the method described by Horsch et al. (Science, 223:496-498, 1984) with the modifications of Fisher and Guiltinan (PlantMol. Biol. Reporter 13: 278-289, 1995). After incubation, the leaf diskswere transferred to plates with regeneration and selection medium (MSSmedium with IAA 0.2 mg/l, 6-BAP 2.2 mg/l, carbenicillin 400 mg/l andkanamycin 100 mg/l), and the buds that appeared were transferred to therooting medium (MSS medium with IAA 0.2 mg/l, carbenicillin 200 mg/l andkanamycin 100 mg/l). The regenerated plants were transferred to potswith peat: vermiculite (1:1) where they were kept until they producedseeds. The culture conditions during the whole process were 12 h oflight and a temperature of 24° C.

[0082] For the production of transgenic plants of A. thaliana theColumbia cv was used. The transformation protocol was followed byinfiltration under vacuum and selection of the plants resistant tokanamycin described by Bechtold et al. (C R Acad. Sci. Paris, Life Sci.,316: 1194-1199, 1993). The strain C58 of A. tumefaciens was used. Theplants resistant to kanamycin were transferred to pots with vermiculite:perlite: peat (1:1:1) and grown in culture chambers at 22° C. inlong-day conditions until the seeds were finally collected.

[0083] In order to obtain transgenic tomato plants, the growth varietyknown as VC82b was used, following the method of Ellul et al., (Teor.Appl. Genet. In press, 2001) that uses cotyledons from germinating seeds(12 days) as starting material and the marker gene nptII to carry outselection of the transformants in a kanamycin medium.

[0084] 1.3.—Histochemical Analysis (GUS) of the Transgenic Plants

[0085] The first generation of transformed plants was submitted tohistochemical analysis of the activity of the b-glucuronidase gene. Thestudied tissues were infiltrated using two vacuum pulses of 5 min in a0.1 M pH 7.0 phosphate buffer solution, 0.5 nM ferricyanide, 0.5 nMferrocyanide, Triton X-100 at 0.1% and 2 mM X-G1cA acid and incubated inthis solution at 37° C. for 16 hours. Afterwards, de-staining wascarried out using successive washes with ethanol at 50°, 70° and 96°.GUS positive zones were identified as those coloured blue. The tissuesthat showed a blue coloration were fixed and included in paraffin (CañasLA et al., Plant J. 6: 597-604, 1994) before being dehydrated, in orderto identify by sections which types of cell in particular wereresponsible for b-glucuronidase activity. The photographs of theunprocessed tissues were taken with an MZ8 lens (Leica) and the tissuesections included in paraffin in an optical microscope Eclipse 600(Nikon).

[0086] Analysis of Transgenic Plants of Arabidopsis thaliana

[0087] The study of the expression of the gene uidA (GUS-intron) wascarried out by means of a histochemical assay of the b-glucuronidaseactivity in first generation tissues of transformed plants resistant tokanamycin. The organs assayed were: flowers, leaves, stems, roots andgerminated seedlings.

[0088] We analysed 26 plants transformed with the pBI101-F3 construct,of which 24 showed specific b-glucuronidase activity and the remainingtwo did not shown any activity in any of the assessed tissues (FIG. 5).Of 19 plants transformed with the pBI101-F 1.5 construct and resistantto kanamycin, only two showed lack of GUS activity. In the remaining 17,blue coloration was observed specifically in anthers. The anther tissuesthat showed GUS activity were the same as those in which the END1 geneof pea is expressed: epidermis, connective, middle layer andendothecium. The staminal filament, the central vascular cylinder,nutritive tissue (tapetum), the pollen mother cells and the adult pollendid not show GUS activity in any case. The plants that were transformedwith the pBI101 plasmid (negative control) did not show GUS activity inany of the analysed tissues.

[0089] No differences were observed in GUS activity between plantstransformed with one or another construct. However, we observevariability in the intensity of coloration between flowers of the sameplant, regardless of the construct used, since we can alreadydistinguish flowers in a certain phase that did not show GUS activityand others in the same phase that did show GUS activity. Possibly, thisphenomenon is due to the different degrees of penetration of thereagents of the assay in some tissues compared with others. Thesimilarity of the results obtained with both constructs indicates thatall regulatory sequences necessary for activity are to be found in thefragment comprised between residues −1531 and −6, although more studywould be necessary to determine the minimum fragment able to maintainpromoter activity.

[0090] 1.5.—Analysis of the Transgenic Plants of Tobacco (Nicotianatabacum)

[0091] The study of the expression of the uidA gene was carried out inthe first generation of plants with the pBI101-F3 construct. The tissuesthat were assayed were: flowers, leaves, stems and roots.

[0092] We analysed 12 plants of tobacco resistant to kanamycin, of which10 showed GUS activity in their anthers and two did not show anyactivity in the tissues assayed (FIG. 6). We found the same variabilityin blue coloration between plants from the same plant as observed in A.thaliana. The plants transformed with the pBI101 plasmid (negativecontrol) did not show b-glucuronidase activity in any of the tissues asexpected.

[0093] 1.6.—Analysis of Transgenic Plants of Tomato (Lycopersicomsculentum)

[0094] 10 different lines of plants resistant to kanamycin wereanalysed, of which, half turned out to be positive when expression ofthe uidA gene in anthers was analysed. Study of the expression of saidgene was carried out in the first generation of plants transformed withthe pBI101-F3 construct. The tissues assayed were: flowers, leaves,stems and roots. As in previous cases, the results were practicallyidentical, observing the expression of the GUS gene only in thosetissues that form the pollen sac of the anther (FIG. 7).

EXAMPLE 2

[0095] Production of androsterile plants of Arabidopsis thaliana usingthe barnase gene under the control of the END1 promoter

[0096] 2.1.—Design of the Construct pBI101-pEND1-barnase/barstar

[0097] The pBI101-F3 construct that contains fragment 2731 bp of theEND1 promoter and the GUS gene, was digested with the BamHI and SacIrestriction enzymes, to give rise to the release of the GUS fragment.The fragment corresponding to the pBI101-pEND1 plasmid with BamHI andSacI termini was isolated on agarose gel using the Quiaex II system(Quiagen). The barnase/barstar fragment (Mariani et al., Nature 347:737-741, 1990; Mariani et al., Nature 357: 384-387, 1992), cloned at theBamHI site of the plasmid pBluescript KS (+), was amplified using theoligos: T7: 5′ TAATACGACTCACTATAGGG 3′, e Inhi II:5′ GCGAGCTCTTAAGAAAGTATGATGGTGATG 3′

[0098] With the first, the splicing site BamHI is maintained at thelevel of the ATG of the barnase gene and the later creates a splicingsite for SacI at the level of the stop codon of the barstar gene. Thefragment product of the PCR reaction was bound to the pGEM-T vector(Promega). Finally, the cloned barnase/barstar insert (918 bp) wasreleased with BamHI and SacI and bound to the pBI101-pEND1 plasmid withBamHI and SacI termini.

[0099] 2.2.—Results and Comments

[0100] The results obtained are shown in FIG. 8. The plants transformedwith the pEND1-BARNASE construct showed how the carpels of their flowersare not fertilised and do not form the corresponding siliques as occursin the control plant. On the other hand, the flowers of the transgenicplants showed the filaments that have not lengthened and at whose endthere appear some very rudimentary structures instead of anthers. Thetotal of 17 transgenic plants resistant to kanamycin recovered showed acomplete ablation of their anthers and therefore, 100% male sterility.

[0101] The studies carried out with a scanning electron microscope (SEM)showed (FIG. 9) that the cell types present in said structures do notcorrespond to those present in the epidermis of an anther, detectingonly those that form the filament or those that are present at the zonewhere the filament joins the anther. These studies were completed withsections of structures in paraffin with subsequent staining thereof withAlcian blue and Safranin to detect cell lines related to the developmentof the pollen. In FIG. 10, we see how small groups of mother cells ofpollen and perhaps of external tapetum (in red) are observed in theinterior of the structures located at the end of the filament. Thesecells seem to have stopped their development on comparing them with theprocess that normally produces an untransformed anther.

[0102] As has already been explained, the barnase/barstar system hasbeen successfully used in the production of plants with male sterilitybecause it is directed towards the anthers of the plant by means of theuse of specific promoters (generally of tapetum). The main disadvantageof this system lies in the use of said promoters, which act too late inthe development of the anther and on a tissue (tapetum) implicated inthe nutrition of the cells that will give rise to the pollen. Thiseffect leads to the production to a certain extent of pollen in someflowers of the plant, causing escapes from the system with thecorresponding loss in effectiveness (<95%).

[0103] Using the promoter of the END1 gene these problems do not existdue to three reasons:

[0104] The expression of BARNASE (RNase) is carried out in very earlydevelopmental phases, leading to complete ablation of the cell linesthat will give rise to the tissues that form the pollen sacs: epidermis,endothecium, connective, middle layer, etc.

[0105] Only the filament is formed at the base of the anther and thisnever lengthens to reach the stigma of the carpel.

[0106] Although the cell lines that will give rise to external tapetumand to pollen mother cells do not experience the effects of BARNASE asthey are not a target for the END1 promoter, they stop in an early phaseof their development as they do not have the support of the remainingtissues of the anther, which would also be fundamental for the processof mature pollen release (dehiscence).

1 5 1 2766 DNA Pisum sativum promoter (1)..(2736) 1 gacttcaaccttattagtga atggacaata aaggttataa gctcctttac tgtgaaagcc 60 caccagtaacatcaccttgc ttatatcatt cagcttcttt ctagtaacat ttggaacgtg 120 tttataacagaaaaaaaccc aaaaactctg aaaagactca cacttttctt atctccagtc 180 cacctctcaaaaggaacaat ttccttcagc ttcttggttg gacacctgtt gagcacatat 240 gctgcagtggcaacagtttc tccccacaaa gtgttaggaa gcttcttctc cttcagcatg 300 ttccttgtcatatcaagcaa agttcggttt tcaacaagac cattatgttg aggagtatat 360 ggatcagtcacttcatgctc aattccattc tctttacaga acttcttgaa ctctgtagag 420 ttatactcacctccaccatc agttctgaga atcttcagaa gtctgaccac tttatttctc 480 agccttgattatgaatttct taaattcagc aaacacctcg tgtttgaatt ttataaggga 540 tacccatgtcatccttgtga actcatccat aaatgacata aagtattatt ccctcctagt 600 gaaaggtttgtaatgggcca cacacataag aatgcactac tcctaaagca tgttttgctc 660 tttgagctacttttgatgaa aatggcagtc ttggttgctt ccctttcatg cacacattac 720 atgacttttttggtttctta attgtaggaa ttccacgtac cagtttcttt gaattcaaat 780 tccctaagctcctaaagttc aaatgaccaa atcttttgtt ccacaactca ctttccttca 840 caacacttgttgcgctaagg cattcagagt ctgcagtttt aacattcgcc ttgaatgttt 900 tactccttccatgttctgac tccataatca acttctgata acagtcatac agcttcaaaa 960 gaatgtcattcatggtaact ggaaatccct tttcaattaa ttgacctaca ctcatcagat 1020 tgctcttcatgccaagaacg taccaagacg ttctgaatta atgcagattt tctattattc 1080 ataatcactctaacattccc cattccttta gcatttagtt acttatcatc agcacatcta 1140 atcttggttttcttcctaga gtcaaaatca accagccatt tcttatttcc agtatgatgg 1200 tttgaacaaccagtgtccat atatcaccag tcttctatag acgcactatc ataactagaa 1260 gccattaatagcacatgttc atcatggtgc tcagatcctt agaatgttca attgctacaa 1320 cgatgtaatcaaactgatga gtaagagatc taagtacctt ctcaatgata ctttcctcat 1380 aaagagtttctccatgcgac ttcatctcat ttgtgatcag aatcactcta gagatgtagt 1440 cagataacttctcattgttc ttcatgctta gattctcata ctgctcacgt agagactgaa 1500 gtttcaccttctacactgat gcatcactat cgtagcacca caccagtctg tctcacacaa 1560 ccttttccgtcattgaatca acgattttct taaacacgtt cacatccaca cactgatgga 1620 tgtagaacaacgcattctga tccttcttcc tcatatcaca ctgagcattt ctttgcgcat 1680 ccgttgcattttctagaagt gaagcataaa cttcgttgat gagatcaaga acatcttgag 1740 caccaaataacacacacatc tgaatcatcc aacgattcca gttgttgtcg tcgaacaatg 1800 gnagcntggtgcacagattc acaacgatat attataantt ttgttttatg aaatttaaga 1860 acaaatttccattattctta aaatgtttac acactgatgt agactgcaaa aggaataaag 1920 atacaatttgttcacaccac tcacttgcgt aaatataagt gagagttaat gagaaatact 1980 aaaataccctctaaaattat gaattaattc taacaatctc taatgttagt ataatccatt 2040 aaacactttgatggcaggta taacaagggt gtaagttagt gtatacatat taggctctta 2100 ttatttttatattatctctg cttttcttct tcatgttctc actaatatga tattatctcc 2160 cttccctaaattatttatat ttattagaaa aagagtttca ttttttaaaa atatattacc 2220 gtaatttttcaaaaaataaa atttaaatat attttataaa aaaattattt aataatttat 2280 ttacattaatgcataaatat aaataaatac tgtcatttaa tatttaacct tttaacaata 2340 aattatatttatttaattca actaatataa gctaagttat ctcatccaac caattaaaaa 2400 gatcatttgaaaataccttt ttatttagtt tgtggcggtt tcaactgtca aaaaaaagga 2460 atttttacgacgatataaat ttaaaccagc aaaaaattga agcagttaag cgaaccaact 2520 catggtatgtggatatattt atctttgtcg tttatatcgg attcgaatct ctataatgat 2580 gaaaaattaatatcaaactt taaataagaa cgtcatttat agagccattt tgggaaacac 2640 atatttcatgtacacgtgat tcgcaaattt ccaataactc tatatatagc cctcctcagt 2700 ttcatgcatttgctcacaac ataaccttcc ttgaattcga tatctaccta agatgacaaa 2760 accagg 27662 910 DNA Pisum sativum CDS (17)..(706) 2 tcgatatcta cctaag atg aca aaacca ggt tac att aat gct gct ttt cgt 52 Met Thr Lys Pro Gly Tyr Ile AsnAla Ala Phe Arg 1 5 10 tca tct ttc aac ggc gaa cgt tac tta ttc atc gatgat aag tat gtg 100 Ser Ser Phe Asn Gly Glu Arg Tyr Leu Phe Ile Asp AspLys Tyr Val 15 20 25 ttg gta gat tat gca ccg gga acc cgc gac gat aag ctctta aac ggg 148 Leu Val Asp Tyr Ala Pro Gly Thr Arg Asp Asp Lys Leu LeuAsn Gly 30 35 40 cct ctt cct ctt cct gct ggg ttt aaa tca ctt gat ggt acagta ttt 196 Pro Leu Pro Leu Pro Ala Gly Phe Lys Ser Leu Asp Gly Thr ValPhe 45 50 55 60 gga acc tac gga gtt gac tgt gcc ttt gac acc gat aac gacgaa gca 244 Gly Thr Tyr Gly Val Asp Cys Ala Phe Asp Thr Asp Asn Asp GluAla 65 70 75 ttc atc ttt tat gag aac ttt act gct ctc ata aac tat gct ccacat 292 Phe Ile Phe Tyr Glu Asn Phe Thr Ala Leu Ile Asn Tyr Ala Pro His80 85 90 act tac aat gac aaa atc atc tcg ggt ccg aag aaa atc tcg gac atg340 Thr Tyr Asn Asp Lys Ile Ile Ser Gly Pro Lys Lys Ile Ser Asp Met 95100 105 ttt cct ttt ttc aaa gga acc gtg ttt gaa aac ggg att gac gct gca388 Phe Pro Phe Phe Lys Gly Thr Val Phe Glu Asn Gly Ile Asp Ala Ala 110115 120 ttc agg tca act aag gag aaa gaa gtt tat tta ttc aaa gga gac ttg436 Phe Arg Ser Thr Lys Glu Lys Glu Val Tyr Leu Phe Lys Gly Asp Leu 125130 135 140 tat gct cgt ata gac tat gga aaa aac tat ctg gtt caa agt atcaag 484 Tyr Ala Arg Ile Asp Tyr Gly Lys Asn Tyr Leu Val Gln Ser Ile Lys145 150 155 aac att agc act ggg ttc cct tgt ttc act gga acc gtc ttt gaaaat 532 Asn Ile Ser Thr Gly Phe Pro Cys Phe Thr Gly Thr Val Phe Glu Asn160 165 170 gga gtg gat gct gct ttt gct tct cac agg acc aat gaa gca tacttt 580 Gly Val Asp Ala Ala Phe Ala Ser His Arg Thr Asn Glu Ala Tyr Phe175 180 185 ttc aaa gga gat tac tat gca ctt gtc aag att agc ccg ggc ggaata 628 Phe Lys Gly Asp Tyr Tyr Ala Leu Val Lys Ile Ser Pro Gly Gly Ile190 195 200 gat gac tat att atc ggt ggt gtg aag ccc att ctt gag aat tggcct 676 Asp Asp Tyr Ile Ile Gly Gly Val Lys Pro Ile Leu Glu Asn Trp Pro205 210 215 220 tct ctt cgt ggt ata ata cct cag aaa agt taaatgtggctctctgtgtg 726 Ser Leu Arg Gly Ile Ile Pro Gln Lys Ser 225 230tgtgtgatat catcagtcaa gtatggtatt aagaataaag actattgttg tcgttgttgt 786gtgtttcttt ttcatgttgt ttctagttct taatgtttgc ttatgttgtt catgtgaact 846atgtaatgac atgcactgtg tacgcgcaga gtgaaaataa tatattactg tgtatgttga 906ttac 910 3 230 PRT Pisum sativum 3 Met Thr Lys Pro Gly Tyr Ile Asn AlaAla Phe Arg Ser Ser Phe Asn 1 5 10 15 Gly Glu Arg Tyr Leu Phe Ile AspAsp Lys Tyr Val Leu Val Asp Tyr 20 25 30 Ala Pro Gly Thr Arg Asp Asp LysLeu Leu Asn Gly Pro Leu Pro Leu 35 40 45 Pro Ala Gly Phe Lys Ser Leu AspGly Thr Val Phe Gly Thr Tyr Gly 50 55 60 Val Asp Cys Ala Phe Asp Thr AspAsn Asp Glu Ala Phe Ile Phe Tyr 65 70 75 80 Glu Asn Phe Thr Ala Leu IleAsn Tyr Ala Pro His Thr Tyr Asn Asp 85 90 95 Lys Ile Ile Ser Gly Pro LysLys Ile Ser Asp Met Phe Pro Phe Phe 100 105 110 Lys Gly Thr Val Phe GluAsn Gly Ile Asp Ala Ala Phe Arg Ser Thr 115 120 125 Lys Glu Lys Glu ValTyr Leu Phe Lys Gly Asp Leu Tyr Ala Arg Ile 130 135 140 Asp Tyr Gly LysAsn Tyr Leu Val Gln Ser Ile Lys Asn Ile Ser Thr 145 150 155 160 Gly PhePro Cys Phe Thr Gly Thr Val Phe Glu Asn Gly Val Asp Ala 165 170 175 AlaPhe Ala Ser His Arg Thr Asn Glu Ala Tyr Phe Phe Lys Gly Asp 180 185 190Tyr Tyr Ala Leu Val Lys Ile Ser Pro Gly Gly Ile Asp Asp Tyr Ile 195 200205 Ile Gly Gly Val Lys Pro Ile Leu Glu Asn Trp Pro Ser Leu Arg Gly 210215 220 Ile Ile Pro Gln Lys Ser 225 230 4 230 PRT Pisum sativum 4 MetThr Lys Pro Gly Tyr Ile Asn Ala Ala Phe Arg Ser Ser Phe Asn 1 5 10 15Gly Glu Arg Tyr Leu Phe Ile Asp Asp Lys Tyr Val Leu Val Asp Tyr 20 25 30Ala Pro Gly Thr Arg Asp Asp Lys Leu Leu Asn Gly Pro Leu Pro Leu 35 40 45Pro Ala Gly Phe Lys Ser Leu Asp Gly Thr Val Phe Gly Thr Tyr Gly 50 55 60Val Asp Cys Ala Phe Asp Thr Asp Asn Asp Glu Ala Phe Ile Phe Tyr 65 70 7580 Glu Asn Phe Thr Ala Leu Ile Asn Tyr Ala Pro His Thr Tyr Asn Asp 85 9095 Lys Ile Ile Ser Gly Pro Lys Lys Ile Ser Asp Met Phe Pro Phe Phe 100105 110 Lys Gly Thr Val Phe Glu Asn Gly Ile Asp Ala Ala Phe Arg Ser Thr115 120 125 Lys Glu Lys Glu Val Tyr Leu Phe Lys Gly Asp Leu Tyr Ala ArgIle 130 135 140 Asp Tyr Gly Lys Asn Tyr Leu Val Gln Ser Ile Lys Asn IleSer Thr 145 150 155 160 Gly Phe Pro Cys Phe Thr Gly Thr Val Phe Glu AsnGly Val Asp Ala 165 170 175 Ala Phe Ala Ser His Arg Thr Asn Glu Ala TyrPhe Phe Lys Gly Asp 180 185 190 Tyr Tyr Ala Leu Val Lys Ile Ser Pro GlyGly Ile Asp Asp Tyr Ile 195 200 205 Ile Gly Gly Val Lys Pro Ile Leu GluAsn Trp Pro Ser Leu Arg Gly 210 215 220 Ile Ile Pro Gln Lys Ser 225 2305 1561 DNA Pisum sativum promoter (1)..(1561) 5 acaaccagtg tccatatatcaccagtcttc tatagacgca ctatcataac tagaagccat 60 taatagcaca tgttcatcatggtgctcaga tccttagaat gttcaattgc tacaacgatg 120 taatcaaact gatgagtaagagatctaagt accttctcaa tgatactttc ctcataaaga 180 gtttctccat gcgacttcatctcatttgtg atcagaatca ctctagagat gtagtcagat 240 aacttctcat tgttcttcatgcttagattc tcatactgct cacgtagaga ctgaagtttc 300 accttctaca ctgatgcatcactatcgtag caccacacca gtctgtctca cacaaccttt 360 tccgtcattg aatcaacgattttcttaaac acgttcacat ccacacactg atggatgtag 420 aacaacgcat tctgatccttcttcctcata tcacactgag catttctttg cgcatccgtt 480 gcattttcta gaagtgaagcataaacttcg ttgatgagat caagaacatc ttgagcacca 540 aataacacac acatctgaatcatccaacga ttccagttgt tgtcgtcgaa caatggnagc 600 ntggtgcaca gattcacaacgatatattat aanttttgtt ttatgaaatt taagaacaaa 660 tttccattat tcttaaaatgtttacacact gatgtagact gcaaaaggaa taaagataca 720 atttgttcac accactcacttgcgtaaata taagtgagag ttaatgagaa atactaaaat 780 accctctaaa attatgaattaattctaaca atctctaatg ttagtataat ccattaaaca 840 ctttgatggc aggtataacaagggtgtaag ttagtgtata catattaggc tcttattatt 900 tttatattat ctctgcttttcttcttcatg ttctcactaa tatgatatta tctcccttcc 960 ctaaattatt tatatttattagaaaaagag tttcattttt taaaaatata ttaccgtaat 1020 ttttcaaaaa ataaaatttaaatatatttt ataaaaaaat tatttaataa tttatttaca 1080 ttaatgcata aatataaataaatactgtca tttaatattt aaccttttaa caataaatta 1140 tatttattta attcaactaatataagctaa gttatctcat ccaaccaatt aaaaagatca 1200 tttgaaaata cctttttatttagtttgtgg cggtttcaac tgtcaaaaaa aaggaatttt 1260 tacgacgata taaatttaaaccagcaaaaa attgaagcag ttaagcgaac caactcatgg 1320 tatgtggata tatttatctttgtcgtttat atcggattcg aatctctata atgatgaaaa 1380 attaatatca aactttaaataagaacgtca tttatagagc cattttggga aacacatatt 1440 tcatgtacac gtgattcgcaaatttccaat aactctatat atagccctcc tcagtttcat 1500 gcatttgctc acaacataaccttccttgaa ttcgatatct acctaagatg acaaaaccag 1560 g 1561

We claim:
 1. A nucleotide sequence regulating the specific expression inanthers of a gene selected from: a) a nucleotide sequence comprising thenucleotide sequence shown in SEQ ID NO 1; b) a fragment of any of saidSEQ ID NO 1 that maintains the capacity to regulate the specificexpression in the anther; and c) a nucleotide sequence that issubstantially analogous to the nucleotide sequence defined in a) or inb).
 2. A regulatory sequence of nucleotides in accordance with claim 1,comprising the nucleotide sequence shown in SEQ ID NO
 4. 3. A DNAconstruct comprising a nucleotide sequence in accordance with any ofclaims 1 or 2 and a coding nucleotide sequence of a peptide,polypeptide, protein, activity or RNA.
 4. A recombinant vectorcomprising a nucleotide sequence in accordance with any of claims 1 or2, or a DNA construct in accordance with claim
 3. 5. A transformed cellthat comprises a nucleotide sequence in accordance with any of claims 1or 2, or a DNA construct according to claim 3, or a recombinant vectorin accordance with claim
 4. 6. A transgenic cell comprising, inserted inits genome, a nucleotide sequence in accordance with any of claims 1 or2, or a DNA construct according to claim 3, or a recombinant vector inaccordance with claim
 4. 7. A transgenic plant comprising at least atransgenic cell in accordance with claim
 6. 8. A transgenic plant inaccordance with claim. 7, at least comprising a transgenic cell thatcontains a DNA construct according to claim 3, wherein said codingnucleotide sequence codes a peptide, polypeptide, protein or activity orRNA that is cytotoxic and whose expression induces the ablation of theanther, and said coding sequence is controlled by a nucleotide sequenceaccording to any of claims 1 or
 2. 9. A transgenic plant according toclaim 7, selected from among monocotyledoneous and dicotyledoneous. 10.The use of a transgenic plant in accordance with any of claims 7 to 9 toproduce hybrid seeds.
 11. A method for producing androsterile plants,which comprises transforming a plant with a DNA construct according toclaim 3, said DNA construct comprising a coding sequence of promoteractivity of the sterility in plants, controlled by a nucleotide sequenceaccording to any of claims 1 or 2, so that said expression of saidcoding sequence produce the ablation of the anthers.
 12. A method forrestoring the fertility of an androsterile plant that comprisestransforming said androsterile plant with a DNA construct according toclaim 3, said construct comprising a coding sequence of an activity thatreverses or restores the fertility that inhibits the activity ofablation of the anthers, controlled by a nucleotide sequence accordingto any of claims 1 or 2.